Coronary artery bypass grafting (CABG) with autologous conduits effectively alleviates ischemic heart disease, but long-term graft patency is problematic. Graft failure is primarily attributable to intimal hyperplasia (IH), the process by which vascular smooth muscle cells (VSMCs) migrate, proliferate, and deposit excessive extracellular matrix (ECM) resulting in neointima formation. IH can occlude the lumen and is prone to rapid development into advanced graft disease, negating the surgical benefit. We have identified MAPKAP kinase II (MK2) as a potential target for pharmacological intervention for preventing vascular graft IH. MK2 is activated through a p38 mitogen activated kinase (MAPK) pathway that is triggered by the physical and biochemical stresses that VSMCs in the graft experience during transplant. Activated MK2, in turn, phosphorylates heat shock protein 27 (HSP27), a known downstream mediator of pathological VSMC behavior in IH. Because p38 MAPK functions are diverse, p38 inhibitors lead to nonspecific side effects. Thus, MK2 is a logical target for inhibiting a proximal trigger of IH, and the central hypothesis of this proposal is that efficient pharmacological inhibition of MK2 will prevent IH. Pharmaceutical companies'efforts to develop a small molecule MK2 inhibitor have been unsuccessful. Peptide inhibitors hold promise as an alternative with greater specificity and reduced toxicity, but barriers against intracellular delivery hinder their use. To test our hypothesis, a novel, "smart" polymer vehicle for intracellular delivery of a peptidic MK2 inhibitor (MK2i) is proposed. Preliminary data have shown a reduction in IH in human saphenous vein samples delivered MK2i via fusion to a cell penetrating peptide (CPP-MK2i). However, our mechanistic studies support the notion that CPP-mediated delivery suffers from compromised cytoplasmic bioactivity due to internalization into and sequestration within intracellular vesicles. The overall goal of this proposal is to overcome this cytoplasmic delivery barrier and to optimize MK2 inhibition using a CPP-internalized "smart" polymer (CISP) for MK2i peptide delivery (CISP-MK2i). The proposed "smart" polymer carrier will "sense" the acidic environment in the endosomes, which will trigger a sharp polymer transition into a more hydrophobic, membrane disruptive state that releases CISP-MK2i into the cytoplasm. Furthermore, CISP-MK2i has been designed with reducible attachments between MK2i and the "smart" polymer backbone, and upon reaching the cytoplasm, "cell- demanded" MK2i release will occur to ensure that the carrier does not sterically hinder MK2i activity. Three aims are outlined in the proposal for CISP-MK2i synthesis and a series of in vitro, ex vivo, and in vivo tests to compare the efficacy of MK2i delivery via CISP-MK2i versus CPP-MK2i. PUBLIC HEALTH RELEVANCE: Coronary artery bypass grafting with autologous conduits effectively alleviates ischemic heart disease, but long-term graft patency is problematic. Graft failure is primarily attributable to intimal hyperplasia (IH), the process by which vascular smooth muscle cells (VSMCs) migrate, proliferate, and deposit extracellular matrix (ECM) into a neointima. MAPKAP kinase II (MK2) activation by cellular stress during transplant is thought to be an important contributor to the pathological VSMC phenotype. Here we propose to develop and test a "smart" polymer-peptide hybrid delivery system capable of efficiently inhibiting MK2 and improving long-term vascular graft performance.